We are responding to PAR-13-375, Nutrigenetics and Nutrigenomics Approaches for Nutrition Research. This amended proposal, supported by a team of well-established investigators in proteomics, metabolomics, gut microbiome, experimental nutrition, and biostatistics, aims to evaluate effects of specific diet patterns on biomarkers of cancer-risk pathways in a controlled feeding study. Diet is a complex exposure in humans, aspects of which are recognized to play a role in the risk of several cancers. To date, the focus of experimental nutrition has been on the impact of specific nutrients or dietary constituents on mechanisms of cancer prevention or treatment, with less attention placed on the response to multiple, concurrent nutrient exposures, as would occur in free-living humans consuming food. Observational studies suggest that we also need to understand the response to dietary patterns to better target public health recommendations for cancer prevention. Dietary constituents have pleiotropic effects and modulate a variety of pathways important in carcinogenesis. Proteomics and metabolomics have the capacity to capture this range of responses. Further, the gut microbiome and gut microbial metabolites have been shown to affect some of these pathways and have been implicated in the risk of several cancers. We will use a carefully designed, well-controlled feeding study to address the following aims: 1) Compare the effects of two distinct dietary patterns fed as controlled diets on proteomic biomarkers of regulatory pathways important in cancer susceptibility; 2) Compare the effects of the two dietary patterns on (a) gut microbial community and the gut metagenome and metatranscriptome and (b) the plasma metabolome; and 3) Explore what diet-induced changes in the metatranscriptome and metabolomics profiles explain the variation in proteomic measures that also changed in response to diet. We will use data and samples from a completed randomized cross-over feeding study, Carbohydrate and Related Biomarkers (CARB), where 80 participants [40 normal weight (BMI 18.5-25.0 kg/m2) and 40 overweight to obese (BMI 28.0-40.0 kg/m2)] consumed two controlled diets for 28 days each. One diet was high in whole grains, fresh fruits and vegetables. The other substituted refined grains for whole grains, and provided carbohydrates from mostly high-glycemic index food sources. All food was provided to participants and energy content and macronutrient composition was identical in both diets. Each participant served as their own control. Fasting blood and stool were collected at the beginning and end of each diet. We will use a novel protein antibody array to measure the plasma proteome, will characterize the gut microbial community using 16S rRNA sequencing, metagenomics and metatranscriptomics, and will measure the plasma metabolome using targeted and global approaches. We will use novel statistical methods for integrating the various omics data and test for associations using state-of-the- art single-marker and pathway-enrichment statistical models which adjust for demographic variation.